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Interaction of the Human Androgen Receptor Transactivation Function with the General Transcription Factor TFIIF

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Interaction of the Human Androgen Receptor Transactivation Function with the General Transcription Factor TFIIF Proc. Natl. Acad. Sci. USA Vol. 94, pp. 8485–8490, August 1997 Biochemistry Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF IAIN J. MCEWAN* AND JAN-ÅKE GUSTAFSSON Department of Biosciences, Novum, Karolinska Institute, S-141 57 Huddinge, Sweden Communicated by Elwood V. Jensen, University of Hamburg, Hamburg, Germany, May 27, 1997 (received for review January 28, 1997) ABSTRACT The human androgen receptor (AR) is a tion factors, and thus the polymerase, to the promoter (re- ligand-activated transcription factor that regulates genes im- viewed in refs. 17–19). This can be achieved by direct contact portant for male sexual differentiation and development. To between the activator and the general transcription factors better understand the role of the receptor as a transcription andyor interactions by means of coactivator proteins (refs. 17 factor we have studied the mechanism of action of the N- and 19–21 and references therein). terminal transactivation function. In a protein–protein inter- In recent years a number of interactions have been described action assay the AR N terminus (amino acids 142–485) between members of the steroid–thyroid hormone receptor selectively bound to the basal transcription factors TFIIF and superfamily and basal transcription factors and co–activator the TATA-box-binding protein (TBP). Reconstitution of the proteins (see ref. 22 and references therein). However, very transactivation activity in vitro revealed that AR142–485 fused to little is known concerning the identity of interacting proteins the LexA protein DNA-binding domain was competent to with the human AR. To better understand the mechanism of activate a reporter gene in the presence of a competing DNA gene regulation by the human AR we have screened a panel of template lacking LexA binding sites. Furthermore, consistent general transcription factors for binding to the receptor N- with direct interaction with basal transcription factors, ad- terminal transactivation domain and have reconstituted recep- dition of recombinant TFIIF relieved squelching of basal tor-dependent activation under cell-free conditions. A region transcription by AR142–485. Taken together these results sug- of the N terminus, containing the major transactivation activ- gest that one mechanism of transcriptional activation by the ity, is capable of recruiting the general transcription machinery AR involves binding to TFIIF and recruitment of the tran- to a target promoter and shows selective binding to the general scriptional machinery. transcription factor TFIIF. The androgen receptor (AR) is a member of the steroid– MATERIALS AND METHODS thyroid hormone receptor superfamily and mediates the ef- fects of the male sex hormones testosterone and dihydrotest- AR Expression Constructs. The DNA sequence coding for osterone (for review see ref. 1). Mutations in the receptor amino acids 142–485 of the human AR N terminus was proteins have been identified in disorders of male sexual amplified by using the Expand Long Template PCR system differentiation (2, 3), X-chromosome-linked spinal bulbar (Boehringer Mannheim) from plasmid pSVARo (a gift from muscular atrophy (4, 5), prostatic carcinoma (6, 7), and male A. O. Brinkmann, Erasmus University, Rotterdam, The Neth- breast cancer (8). Although there is good evidence that the AR erlands; see ref. 9). The primers used were ARN142, 59- binds to DNA response elements and activates gene expres- GCGCGCAGATCTCTGCCGCAGCAGCTGCCAGC-39, sion, the underlying mechanisms are not well understood. The and ARC485, 59-GCGCGCGGATCCGCTTTCCTGGC- C-terminal steroid-binding domain and the central DNA- CCGCCAGCCCC-39. The PCR product was cleaved with binding domain show significant homology between ARs of BamHI and BglII and ligated into pET-19bm (23) previously different species and also with other members of the nuclear digested with BamHI. The resulting plasmid, pET-AR4, ex- receptor superfamily (ref. 1 and references therein). In con- pressed the AR142–485 domain fused to an N-terminal histidine trast, the N terminus of the protein is more divergent and is tag. The insert was checked for orientation and sequenced characterized by homopolymer tracts of glutamine, glycine, (Dye terminator cycle sequencing, Perkin–Elmer). The ex- and proline residues (ref. 9 and references therein). Regions pression plasmid pET-AR4-Lex was constructed by ligating a within the N terminus of the human and rat receptors impor- fragment encoding the LexA DNA-binding domain (LexADBD, tant for transactivation have been delineated by deletion amino acids 1–87) into the regenerated BamHI site, 39 of the analysis (10–13), the use of fusion proteins (14), and point AR sequence in pET-AR4. mutations (15). These studies have highlighted the region Expression and Purification of Recombinant Proteins. between amino acids 142 and 370 (numbering for the human AR142–485 with or without LexADBD was expressed in Esche- receptor), although sequences both N-terminal and C-terminal richia coli strain BL21 plys by isopropyl b-D-thiogalactoside of this region appear to play an important role in the full (IPTG; 1 mM) induction, and the recombinant proteins were activity of the wild-type AR andyor in promoter specific purified from the soluble fraction by Ni21–nitrilotriacetate activity (see ref. 14). (NTA) affinity chromatography. The bound protein was eluted Transcription of mRNA coding genes involves the concerted with 200 mM imidazole and dialyzed against 25 mM Hepes, pH action of RNA polymerase II and a set of at least five general 7.6y100 mM sodium acetatey1mMDTTy0.01% Nonidet P-40. transcription factors (see refs. 16–19 for recent reviews). One Recombinant yeast TATA-box-binding protein (TBP) and mechanism by which gene regulatory proteins are thought to human TFIIF (RAP30 and RAP74) were expressed in bacteria function is by recruiting one or more of the general transcrip- Abbreviations: AR, androgen receptor; AR142–485, AR residues 142– The publication costs of this article were defrayed in part by page charge 485; LexADBD, LexA DNA-binding domain (residues 1–87); TBP, TATA-box-binding protein; SRF, serum response factor. payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed at present address: accordance with 18 U.S.C. §1734 solely to indicate this fact. Department of Molecular and Cell Biology, University of Aberdeen, © 1997 by The National Academy of Sciences 0027-8424y97y948485-6$2.00y0 Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, PNAS is available online at http:yywww.pnas.org. Scotland, United Kingdom. e-mail: [email protected]. 8485 Downloaded by guest on October 2, 2021 8486 Biochemistry: McEwan and Gustafsson Proc. Natl. Acad. Sci. USA 94 (1997) and partially purified as described previously (24). Protein concentrations were measured against BSA standards using the Bradford reagent (Bio-Rad). Protein–Protein Interaction Assay. The microtiter plate interaction assay was essentially as described previously (23, 24). Briefly, AR142–485 or BSA control in binding buffer [20 mM Hepes, pH 7.6y10% (volyvol) glyceroly100 mM KCly0.2 mM EDTAy5 mM MgCl2y5 mM 2-mercaptoethanol] were allowed to adsorb to the surface of a scintillation-microtiter plate (Wallac, Oy, Finland). Unoccupied surfaces were sub- sequently blocked with binding buffer containing 5 mgyml BSA, and the wells were incubated with binding buffer con- taining 1 mgyml BSA and radiolabeled human basal transcrip- tion factors, synthesized in a rabbit reticulocyte lysate system (Promega). After extensive washing with binding buffer 1 1 mgyml BSA, the bound radioactivity was measured directly in a micro b counter (Wallac, Oy, Finland), and bound proteins were recovered in SDS sample buffer. In Vitro Transcription and Squelching Assays. Preparation of yeast nuclear extracts for in vitro transcription, together with reporter genes and reaction conditions have all been described in detail previously (24–26). RESULTS The N-Terminal Region of the Human AR Contacts Basal Transcription Factors. The N terminus of the AR has been shown to contain a complex transactivation function made up of multiple regions (see Introduction). In an attempt to understand the mechanism by which the AR activates tran- scription, a panel of basal transcription factors was screened for interactions with the receptor transactivation domain. A FIG.2. (A) Screening of 35S-labeled human general transcription factors for binding to AR142–485. The measured radioactivity is plotted relative to wells containing BSA only, which was set at 1. The binding of TBP to the C-terminal transactivation domain of the herpes simplex viral activator protein VP16 is shown for comparison. The results represent the mean 6 SD of at least four observations from two or more independent experiments, except TFIIH, subunits where the mean of two observations only is shown. (B) SDSyPAGE analysis of the bound RAP74 subunit of TFIIF to AR142–485. The input represents 5% of the material incubated per well. polypeptide containing amino acids 142–485 of the human receptor was expressed and purified by metal chelation chro- matography (Fig. 1). The purified protein was allowed to adsorb onto the surface of a microtiter plate and incubated with 35S-labeled basal transcription factors TFIIB, TBP, TFIIEa and -b, TFIIF (RAP30 and RAP74), and two of the subunits of TFIIH (p44 and p62). AR142–485 interacted selec- tively with the RAP74
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